Ricarda A. Steinbrecher, PhD
Econexus
Oxford, 6 January 2016 r.steinbrecher@econexus.info

– BioCAD software,
– Robotic cloning,
– Metabolic modelling,
– Protein engineering,
– DNA databases and registries,
– Part and device libraries,
– Regulatory circuits,
– DNA synthesis,
– Gene and genome assembly,
– Genome editing: MAGE, CRISPR, TALENs, zinc fingers **
– Epigenetic engineering **
• Microscopy, Molecular profiling

- designing and controlling biological processes
- making organisms perform according to design
•
Microorganisms
• Plants
• Animals
• Human
of the outcome of our doing & intervention on all levels
?
the dream of the realisation of precision invites a mechanistic approach and mindset
(as we have insufficient knowledge)

Princeton School of Engineering and Applied Science www.princeton.edu/cbe/people/data/j/javalos/profile/

under scrutiny by EC (are they GMOs??):
1) * Zinc Finger Nuclease Technology (ZFN-1/2/3)
2) * Oligonucleotide Directed Mutagenesis (ODM)
3) * Cisgenesis/Intragenesis
4) RNA-dependent DNA methylation (RdDM);
5) Grafting (onto a GMO rootstock);
6) Reverse Breeding (RB);
7) Agro-infiltration (both Agroinfiltration ‘sensu stricto’ & Agro-inoculation)
(8 Synthetic genomics ) plus other NBTs mentioned by industry and interest groups: eg: MAS (marker assisted selection) not GM !

distributed by EuropaBio

Metabolism is the set of chemical reactions that occur in a cell, which enable it to keep living, growing and dividing.
Metabolic processes are usually classified as: catabolism - obtaining energy and reducing power from nutrients. anabolism - production of new cell components, usually through processes that require energy and reducing power obtained from nutrient catabolism.

ct: glycolysis - glucose oxidation in order to obtain ATP cb: citiric acid cycle (
Kreb’s cycle): acetyl-CoA oxidation in order to obtain GTP and valuable intermediates. bl: AA degradation

metabolic map
Organism = chemical factory
 making different compounds according to design
 utilise “precursors”
 Change plumbing (genes)
 “Petrol” in …. product out

cells nucleus chromosome
DNA gene
A
T
A
T
C
G
T
A
C G
G C
T
A
Basepairs: A-T & C-G ( nucleotides )

“The New Genetics”
3. In many cases, more than one RNA/protein is produced from a given gene.
DNA transcription
RNA mRNA processing (alterations, splicing)
3D Protein translation protein

promoter Gene for trait (Bt toxin) end
Regulatory sequence: on/off switch often CaMV (virus)
Coding sequence of a gene e.g. pat or bar gene for herbicide resistance from soil bacteria
Regulatory sequence: Termination signal e.g. from pea
Plasmid backbone DNA, superfluous genetic material

?
Particle bombardment
Agrobacterium used as “shuttle”
No control of where the gene will insert itself:
• Random integration
• Imprecision
(incl. superfluous DNA)
• 100 -1000s of Mutations
(Sala et al. 2000, Wang et al. 1996, Labra et al 2001)

Targeted ‘break’ of both strands of
DNA in a chromsome
Special enzyme

1. CRISPR/Cas
2. ZFN (zinc finger nucleases)
3. Tales/Talens (transcription activator–like effector nucleases)
4. Meganucleases
9/35

ACGTTTTAACGTTTACGGGT … …
A C G T
T
C
G
G G
A C G T T T A A C G T
T
A
A
“Computer, give me 1000 variants” – put into 1000 organisms

Two separate breaks result in large deletion of material
Material
‘deleted’
6/35

the double-stranded break results in degradation of both strands by endogenous enzymes, enlarging the area of damage the damage can be repaired by rejoining (leaving a deletion or other change), by a template-directed repair (that may change function) or a template-directed insertion

Off-target effects (due to non-specific binding to nontarget DNA): off-target mutations in the genome. These mutations can a) if in the coding sequence, result in changes of function of proteins, or b) if in regulatory sequences, result in changes in the expression of genes, such as increased presence of plant toxins, or absence of proteins important for nutrition, plant defence or disease resistance, increased presence of allergens.
- Integration of added DNA or oligonucelotides into the genome
- Impacts of the genetic engineering processes
(mutations)
Steinbrecher, R (2015). Genetic Engineering in Plants and the “New Breeding
Techniques” (NBTs) - Inherent risks and the need to regulate.

The idea of precision is based on that one knows what one is doing – here this is not the case.
Knowledge & precision at the level of nucleotides is only the bottom layer.
What is missing is the contextualisation into
• the genome
• the epigenetic landscape
• the organism
• the ecosystems
• the socio-economic conditions that differ around the world
Precision around nucleotides gives a false sense of predictability and safety – there is no data to support such extrapolations.

Problem for maize yield:
Stem borer (eg chilo partellus moth) effect a third of the region’s maize crop.
Scientists of the Mbita Point Research Station,
Lake Victoria, Kenya came up with a simple solution:
• Pull : Napier grass (up to 70% yield increase).
• Push : Desmodium , a common l egume “weed” species, repels the stem borer
Secondary benefits:
Desmodium enriches soil with nitrates, prevents soil erosion during rains and suppresses the growth of Striga weed, a parasitic plant causing
US$ 10 billion yield loss/year (effecting 100 million Africans).
• Napier grass can be used and sold as fodder.

Locally researched Solutions already Exist

“Biomass doesn’t cut it.. Carbohydrates are not a substitute for oil. I was wrong in that and I admit it. That will never replace oil because the economics don ’t work.
You can’t take carbohydrates and convert them into hydrocarbons economically”
Alan Shaw (formerly
Codexis)